BIOLOGICAL-ACTIVITY OF RECOMBINANT FACTOR-VIII VARIANTS LACKING THE CENTRAL B-DOMAIN AND THE HEAVY-CHAIN SEQUENCE LYS713-ARG740 - DISCORDANT IN-VITRO AND IN-VIVO ACTIVITY

Recombinant factor VIII variants with overlapping deletions spanning t he region Lys713-Ile1668 have been expressed in mammalian cells, and a nalysed for biological activity both in vitro and in vivo. Two distinc t assay systems were used to measure the activity in vitro. The one-st age coagulation assay served to assess factor VIII procoagulant activi ty while a spectrophotometric assay was used for the quantification of factor VIII cofactor activity in factor IXa-dependent factor X activa tion. Deletion of the entire B-domain (Ser741-Arg1648) resulted in a p rotein with similar procoagulant and cofactor activity. In contrast, f actor VIII-del(713-1637), which has a deletion that also comprises the heavy-chain sequence Lys713-Arg740, had lost factor VIII procoagulant activity while factor VIII cofactor activity was retained. This funct ional inconsistency was further addressed by comparing purified factor VIII-del(713-1637) with factor VIII-del(868-1562), a mutant with norm al in vitro activity. Kinetic studies of factor Xa formation revealed that higher concentrations of thrombin were required to develop the co factor activity from factor VIII-del(713-1637) than needed for factor VIII-del(868-1562) or plasma factor VIII. The physiological significan ce of this finding was assessed in dogs with haemophilia A. Both delet ion mutants were similar to plasma factor VIII with regard to binding to von Willebrand factor and half-life and recovery. Employing the cut icle bleeding time model, factor VIII-del(868-1562) was found to be in distinguishable from plasma factor VIII, whereas factor VIII-del(713-1 637) was less effective. The increased thrombin-resistance of factor V III-del(713-1637) thus limits both procoagulant activity and haemostat ic efficacy in cuticle bleeding. These observations suggest that the h eavy-chain sequence LyS713-Arg740, although dispensable for factor VII I cofactor function per se, is involved in the proteolytic activation of factor VIII both in vitro and in vivo.